Aptamers and diagnostic methods for detecting the EGF receptor

ABSTRACT

The present invention provides aptamers that specifically bind to the EGF receptor in a sample, and diagnostic and analytical methods using those aptamers. In some embodiments, the aptamers include a 3′ cap. In some embodiments, the 3′ cap is an inverted deoxythymidine. In some embodiments the aptamers include a spacer and at least one moiety selected from the group consisting of binding pair member and a detectable label, wherein the spacer is attached to the 5′-end of the aptamer and the moiety is attached the 5′ end of the spacer. In some embodiments the spacer is hexaethylene glycol. In some embodiments, the binding pair member biotin. In some embodiments the detectable label is a fluorophore.

FIELD OF THE INVENTION

The invention relates to the field of cancer diagnosis and treatment.Specifically, the invention provides compositions and methods forbinding the EGF receptor when it is not bound to a therapeutic molecule,as well as methods of determining cancer resistance to therapeuticmolecules.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedsimply as an aid in understanding the invention and is not admitted todescribe or constitute prior art to the invention.

The Epidermal Growth Factor receptor (EGFR; also known as HER1 orErbB-1) is a transmembrane tyrosine kinase receptor expressed onepithelial cells that is strongly linked to several cancers, includingbut not limited to head and neck squamous cell carcinoma (HNSCC),non-small cell lung cancer (NSCLC), colorectal cancer (CRC), breastcancer, pancreatic cancer, and brain cancer. The EGFR is shed in bothhealthy patients and cancer patients and is detectable in their serum.Serum levels of EGFR in cancer patients are known to vary from EGFRlevels measured in healthy controls.

The therapeutic antibodies cetuximab (Erbitux™) and panitumumab(Vectibix™) were developed for use as targeted therapies that recognizeand inhibit the extracellular domain of EGFR. Cetuximab is approved bythe FDA for use as an intravenous treatment for head and neck cancer andcolorectal carcinoma, while panitumumab is approved by the FDA for useas a treatment for colorectal cancer.

Some patients have cancer that is resistant or becomes resistant totreatment with cetuximab or panitumumab (see Carney, Expert Rev MolDiagn. 2007 May; 7(3):309-19; Wheeler, et al., Oncogene. 2008 Jun. 26;27(28): 3944-3956; Bardelli, et al., J Clin Oncol. 2010 Mar. 1;28(7):1254-61). Therefore it is desirable for doctors to monitor ongoingtherapy to determine whether cancer cell resistance to the therapeuticantibodies is increasing, and modify the treatment accordingly. It isalso desirable for doctors to determine whether all of the soluble EGFRis bound to the therapeutic antibodies.

SUMMARY OF THE INVENTION

The claimed invention provides aptamers that bind free EGFR in a sample,as well as methods of determining the amount of free EGFR in a sampleand methods of optimizing therapeutic efficacy for treatment of cancer.

In one aspect, the claimed invention provides aptamers including thestructure defined by SEQ ID NO: 1. In some embodiments, the aptamersinclude a 3′ cap. In some embodiments, the 3′ cap is an inverteddeoxythymidine. In some embodiments the aptamers include a spacer and atleast one moiety selected from the group consisting of a binding pairmember and a detectable label, wherein the spacer is attached to the5′-end of the aptamer and the moiety is attached to the 5′ end of thespacer. In some embodiments the spacer is hexaethylene glycol. In someembodiments, the binding pair member is biotin. In some embodiments thedetectable label is a fluorophore. In some embodiments the aptamersinclude the structure defined by SEQ ID NO: 4. In some embodiments theaptamers include the structure defined by SEQ ID NO: 5. In someembodiments the aptamers include the structure defined by SEQ ID NO: 6.In some embodiments the aptamers include the structure defined by SEQ IDNO: 7. In some embodiments the aptamers include the structure defined bySEQ ID NO: 8. In some embodiments the aptamers include the structuredefined by SEQ ID NOS: 1 to 55.

In another aspect, the claimed invention provides methods fordetermining the amount of free Epidermal Growth Factor receptor (EGFR)in a sample including: (a) contacting a sample with an aptamer thatspecifically binds to free EGFR under conditions that allow binding ofthe aptamer to the free EGFR in the sample to form an aptamer-EGFRcomplex; (b) determining the amount of the free EGFR bound by theaptamer; and (c) relating the amount of free EGFR bound by the aptamerto the amount of free EGFR in the sample. In some embodiments theaptamer includes the structure defined by SEQ ID NO: 1. In someembodiments the aptamer includes the structure defined by SEQ ID NO: 4.In some embodiments the aptamers include the structure defined by SEQ IDNO: 5. In some embodiments the aptamers include the structure defined bySEQ ID NO: 6. In some embodiments the aptamers include the structuredefined by SEQ ID NO: 7. In some embodiments the aptamers include thestructure defined by SEQ ID NO: 8. In some embodiments the aptamersinclude the structure defined by SEQ ID NOS: 1 to 55. In someembodiments the aptamer includes a first binding pair member and isimmobilized on a first solid support via a second binding pair memberthat specifically binds to the first binding pair member. In someembodiments, the sample is contacted with the aptamer prior toimmobilization on the first solid support. In some embodiments thesample is contacted with the aptamer after immobilization on the firstsolid support. In some embodiments, the amount of free EGFR isdetermined by determining the amount of EGFR immobilized on the firstsolid support. In some embodiments the amount of EGFR is determinedusing an antibody that specifically binds to the immobilized EGFR.

In some embodiments the methods further include: (i) attaching a thirdbinding pair member to the EGFR; (ii) releasing the aptamer-EGFR complexfrom the solid support; (iii) immobilizing the aptamer-EGFR complex on asecond solid support via a fourth binding pair member that specificallybinds to the third binding pair member; and (iv) determining the amountof EGFR immobilized to the second solid support. In some embodiments,the amount of EGFR immobilized on the second solid support is determinedusing an antibody that specifically binds to EGFR. In some embodiments,the methods further include labeling the EGFR with a detectable labeleither before or after immobilization on the second solid support. Insome embodiments, the aptamer further includes a detectable label. Insome embodiments the amount of EGFR immobilized on the solid support isdetermined using flow cytometry. In some embodiments the amount of EGFRimmobilized on the second solid support is determined by releasing theaptamer from the aptamer-EGFR complex and determining the amount of thereleased aptamer. In some embodiments, the amount of the releasedaptamer is determined by amplification of the aptamer.

In yet another aspect, the claimed invention provides methods ofoptimizing therapeutic efficacy for treatment of cancer comprising:determining the amount of free EGFR and total EGFR in a sample from apatient being treated with a therapeutic molecule that binds theextracellular domain of the Epithelial Growth Factor receptor (EGFR);identifying a need to increase the dose of the therapeutic moleculeadministered to the patient when the proportion of free EGFR to totalEGFR in the sample is greater than a predetermined threshold, andidentifying that the dose of the therapeutic molecule does not heed tobe increased when the proportion of free EGFR to total EGFR in thesample is less than a predetermined threshold, wherein the threshold isabout 10%. In some embodiments, the threshold is about 1%; is about 2%;is about 3%; is about 4%; is about 5%; is about 10%; is about 15%; isabout 20%; is about 25%; is about 30%; is about 35%; is about 40%; isabout 45%; is about 50%.

In some embodiments, the patient has an epithelial carcinoma. In someembodiments the epithelial carcinoma is selected from the groupconsisting of a squamous cell carcinoma, an adenocarcinoma, and atransitional cell carcinoma. In some embodiments, the methods furtherinclude relating the amount of free EGFR to the amount of bound EGFR. Insome embodiments the methods further include predicting poor carcinomaresponse to continued administration of the therapeutic molecule if theamount of free EGFR is too high.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, the term “about” in quantitative terms refers to plus orminus 10%. For example, “about 3%” would encompass 2.7-3.3% and “about10%” would encompass 9-11%. Moreover, where “about” is used herein inconjunction with a quantitative term it is understood that in additionto the value plus or minus 10%, the exact value of the quantitative termis also contemplated and described. For example, the term “about 3%”expressly contemplates, describes and includes exactly 3%.

As used herein, the term “aptamer” refers to an oligonucleotide that canconform in three-dimensions to bind another molecule with high affinityand specificity.

As used herein, the term “cap” refers to a moiety attached to the 3′ or5′ end of an aptamer or other nucleic acid that changes the stability ofthe nucleic acid, prevents polymerase elongation of the nucleic acid, orincreases the efficiency of nucleic acid dimer formation. The term“capping” refers to the process of adding a cap.

As used herein, “consensus sequence”, when used in reference to a seriesof related nucleic acids, refers to a nucleotide sequence that reflectsthe most common choice of base at each position in the sequence wherethe series of related nucleic acids has been subjected to intensivemathematical and/or sequence analysis.

As used herein, the term “free EGFR” refers to Epidermal Growth Factorreceptor (EGFR) or a portion thereof that is not bound, eithercovalently or non-covalently, to a therapeutic molecule (e.g., anantagonist). Therapeutic molecules include any exogenous molecule thatbinds EGFR and modifies its biological activity including, for example,a therapeutic antibody (e.g., cetuximab and panitumumab), a smallmolecule, or an aptamer. Free EGFR may be bound to other endogenousand/or non-therapeutic molecules.

As used herein, the term “bound EGFR” refers to Epidermal Growth Factorreceptor (EGFR) that is bound, either covalently or non-covalently, toan exogenously-administered therapeutic molecule. Therapeutic moleculesmay include any molecule that binds EGFR and modifies its biologicalactivity including, for example, a therapeutic antibody, a smallmolecule, or an aptamer.

As used herein the term “binding agent” means a molecule that binds to acognate ligand with high affinity and high specificity. A binding agentis typically used to identify the presence of its cognate ligand and canbe detectably labeled to allow identification. An “EGFR binding agent”means a molecule that binds to EGFR with high affinity and highspecificity. Examples of EGFR binding agents include antibodies,aptamers, and ligands of EGFR. EGFR binding agents include EpidermalGrowth Factor (EGF), Transforming Growth Factor α (TGF α),Heparin-Binding EGF-like Growth Factor (HB-EGF), amphiregulin,betacellulin, epigen, and epiregulin.

As used herein, the term “binding pair” refers to a pair of moleculesthat bind to each other with high affinity and specificity. A “bindingpair member” refers to one molecule of a binding pair. For example,streptavidin and biotin are binding pair members that non-covalentlybind with each other. Other exemplary binding pairs include protein Aand the Fc domain of IgG, Fab region of an antibody and its antigen, anda single oligonucleotide strand and its complement.

As used herein, the term “detectable label” refers to a molecule or acompound or a group of molecules or a group of compounds associated witha nucleic acid or a polypeptide and is used to identify the nucleic acidor the polypeptide. Signals from the detectable label may be detected byvarious means and will depend on the nature of the detectable label.Detectable labels may be isotopes, fluorescent moieties, coloredsubstances, enzymes, enzyme substrates, and the like. Examples of meansto detect detectable label include but are not limited to spectroscopic,photochemical, biochemical, immunochemical, electromagnetic,radiochemical, or chemical means, such as fluorescence,chemifluorescence, or chemiluminescence, or any other appropriate means.

As used herein, the term “amplification” means one or more methods knownin the art for copying a target nucleic acid, thereby increasing thenumber of copies of a selected nucleic acid sequence. Amplification maybe exponential or linear. A target nucleic acid may be either DNA orRNA. The sequences amplified in this manner form an “amplicon.” Whilethe exemplary methods described hereinafter relate to amplificationusing the polymerase chain reaction (“PCR”), numerous other methods areknown in the art for amplification of nucleic acids (e.g., isothermalmethods, rolling circle methods, etc.). The skilled artisan willunderstand that these other methods may be used either in place of, ortogether with, PCR methods.

As used herein, the term “specific hybridization” is an indication thattwo nucleic acid sequences share a high degree of complementarity.Specific hybridization complexes form under permissive annealingconditions and remain hybridized after any subsequent washing steps.Permissive conditions for annealing of nucleic acid sequences areroutinely determinable by one of ordinary skill in the art and mayoccur, for example, at 65° C. in the presence of about 6×SSC. Stringencyof hybridization may be expressed, in part, with reference to thetemperature under which the wash steps are carried out. Suchtemperatures are typically selected to be about 5° C. to 20° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength and pH) at which 50% of the target sequence hybridizes toa perfectly matched probe. Equations for calculating Tm and conditionsfor nucleic acid hybridization are known in the art.

As used herein, the term “sample” means any tissue or body fluid thatcan be isolated from the body of an individual. For example, a samplemay include a tissue biopsy, blood, plasma, serum, bile, saliva, urine,tears, perspiration, and the like.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of a detection method using an aptamerto bind and detect a molecule.

FIG. 2 shows a schematic of an aptamer construct. Details are discussedin Example 1.

FIG. 3 shows a graph of the fluorescent intensity from a Luminex xTag™assay for serum samples spiked with successively decreasing amounts ofeither cetuximab or panitumumab. Data was generated by SomaLogic®,Boulder, Co., USA. Details are discussed in Example 7.

FIGS. 4A and 4B show tables providing sequence data and K_(d) data forseveral aptamers to EGFR (as known as ERBB 1). Aptamers 2677-1_0 through26677-1_24 are listed in the sequencing listing as SEQ ID NOS: 9-33,respectively.

FIG. 5 illustrates C-5 modified pyrimidines which may be used in theaptamers described herein.

FIG. 6 illustrates the work flow of the aptamer assay.

FIG. 7 illustrates representative results of quantitative PCR ofserially diluted EGFR-captured SOMAmer.

FIG. 8 illustrates recovery studies using varying amounts of purifiedEGFR spiked into sera of 6 healthy individuals. Bars indicate meanvalues of triplicate runs; error bars indicate the standard error of themean.

FIG. 9 illustrates quantitation of EGFR in serum pre-incubated withpanitumumab or cetuximab. Preincubation had little effect on ELISAquantitation but decreased SOMAmer quantitation in a dose-dependentmanner.

FIG. 10 illustrates 21 unique aptamer sequences identified from the 1425sequences isolated from the SELEX process as described in Example 1 whenmultiple copies are taken into account. The sequences are shown 5′ to3′, wherein all T's are 5-(N-benzylcarboxyamido)-2′-deoxyuracilyl(BndU). The aptamers are listed in the sequencing listing as SEQ ID NOS:34-54 as indicated in the figure.

FIG. 11 illustrates an aptamer consensus frequency table for aptamerconsensus sequence SEQ ID NO: 55. The table summarizes the consensus asa frequency matrix in (1) unique counts and (20 total copy counts).

DETAILED DESCRIPTION OF THE INVENTION

Methods using aptamers to detect EGFR have been described in the art.For example, Esposito, et al. (PLoS One. 2011; 6(9):e24071) describedisolation of an aptamer that binds the extracellular domain of EGFR andinhibits tumor growth in a mouse model of NSCLC. Li, et al. (PLoS One.2011; 6(6):e20299) also describe isolation of an aptamer that binds EGFRin a tumor cell line.

The present invention relates generally to an aptamer that binds withhigh affinity and specificity to free EGFR in patient samples.Specifically, embodiments of the invention include aptamer compositions,as well as methods of detecting free EGFR in a sample, and methods ofdetermining whether a cancer is becoming resistant to treatment with atherapeutic molecule.

Aptamers

Aptamers are oligonucleotides that can form a three-dimensionalstructure that binds another molecule with high affinity andspecificity, similar to that of an antibody. Because of their ability tobind a variety of ligands and the ease and range of options forfunctionalization of nucleic acids, aptamers have been used in place ofantibodies in a variety of therapeutic and diagnostic applications. Forexample, pegaptinib is an FDA approved nucleic acid aptamer developed byMacugen, Inc. that inhibits the activity of vascular endothelial growthfactor to treat neovascularization in age-related macular degeneration.Nucleic acid aptamers have been used as capture moieties, as well asdetection moieties in ELISA-like assays for detecting tularemia antigen(Vivekananda, et al., Lab Invest. 2006 June; 86(6):610-8). Aptamers havealso been used in proteomics assays, affinity chromatography, liquidchromatography, and biosensors (for example, see Iliuk, et al., AnalChem. 2011 Jun. 15; 83(12):4440-52; Lee, et al., Adv Drug Deliv Rev.2010 Apr. 30; 62(6):592-605; Mairal, et al., Anal Bioanal Chem. 2008February; 390(4):989-1007; Ravelet, et al., J Chromatogr A. 2006 Jun. 2;1117(1):1-10).

Aptamer Design

Nucleic acid aptamers are created using an in vitro process known assystematic evolution of ligands by exponential enrichment (SELEX).Briefly, the selection process uses a combinatorial oligonucleotidelibrary in which each oligonucleotide has central region of variablenucleic acids flanked by two regions of fixed sequence. The variableregion of each candidate in the library can be totally or partiallyrandomized. The oligonucleotide library is exposed to a target, such asa protein, under conditions that allow favorable binding betweenoligonucleotide candidates and the target. Following binding, aselective partitioning step is utilized, in which non-binding or poorlybinding oligonucleotides are removed from the mixture, and theoligonucleotide candidates that bound to the target are then removedfrom the target molecule. These selected oligonucleotides are thenenriched using PCR amplification with primers to the fixed regions ofthe oligonucleotide candidates. This process of binding, selectivepartitioning, and amplifying the selected candidate oligonucleotides isrepeated for several rounds. Finally, the selected sequences are clonedand sequenced.

The basic SELEX process has been modified to achieve a number ofspecific objectives. For example, U.S. Pat. App. No. 2009/0098549entitled “SELEX AND PHOTOSELEX” describes methods using the SELEXprocess to generate photoreactive nucleic acid ligands that are capableof both binding and covalently crosslinking to target molecules. U.S.Pat. No. 5,707,796, entitled “Method for Selecting Nucleic Acids on theBasis of Structure” describes the use of the SELEX process inconjunction with gel electrophoresis to select nucleic acid moleculeswith specific structural characteristics, such as bent DNA. U.S. Pat.No. 5,580,737, entitled “High-Affinity Nucleic Acid Ligands ThatDiscriminate Between Theophylline and Caffeine” describes a method foridentifying highly specific nucleic acid ligands able to discriminatebetween closely related molecules, termed Counter-SELEX. U.S. Pat. No.5,567,588, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Solution SELEX” describes a SELEX-based method whichachieves highly efficient partitioning between oligonucleotides havinghigh and low affinity for a target molecule. U.S. Pat. No. 5,496,938,entitled “Nucleic Acid Ligands to HIVRT and HIV-I Rev” describes methodsfor obtaining improved nucleic acid ligands after SELEX has beenperformed. U.S. Pat. No. 5,705,337, entitled “Systematic Evolution ofLigands by Exponential Enrichment ChemiSELEX” describes methods forcovalently linking a nucleic acid ligand to its target.

Nucleotides used in the oligonucleotide library can be modified in anynumber of ways, including modifications of the ribose and/or phosphateand/or base positions. Certain modifications are described in U.S. Pat.No. 5,660,985 entitled “High Affinity Nucleic Acid Ligands ContainingModified Nucleotides”, U.S. Pat. No. 5,428,149 entitled “Method forPalladium Catalyzed Carbon-Carbon Coupling and Products”, U.S. Pat. No.5,580,972 entitled “Purine Nucleoside Modifications by PalladiumCatalyzed Methods”, U.S. Pat. No. 5,719,273 entitled “Nucleosidemodifications by palladium catalyzed methods” all of which areincorporated by reference herein in their entirety. In one embodiment,modifications are those wherein another chemical group is attached tothe 5-position of a pyrimidine or the 2′ position of a sugar. There isno limitation on the type of other chemical group that can beincorporated on the individual nucleotides. In some embodiments, theresulting modified nucleotide is amplifiable or can be modifiedsubsequent to the amplification steps (see, e.g., U.S. Pat. No.6,300,074 entitled “Systematic evolution of ligands by exponentialenrichment: ChemiSELEX”).

Aptamer-Based Assays

Assays that use an aptamer to indirectly detect its cognate ligand aredescribed, for example, in U.S. Pat. No. 7,855,054. In these types ofassays, an aptamer construct is used to capture a ligand on a solidsupport, and contacts a sample that may contain the ligand of theaptamer. The aptamer-ligand complex is partitioned from aptamers thatare not bound to the ligand, and the aptamer portion of theaptamer-ligand complex is then separated from the ligand. The amount ofligand in the sample is then measured indirectly by detecting andquantifying the amount of aptamer that was bound to the sample ligand.Alternatively, the ligand captured from the sample is measured directlyusing an antibody or another aptamer that binds to the ligand.

Dual Capture Assay for EGFR

One embodiment of an aptamer-based assay is illustrated in FIG. 1.Referring to step A of FIG. 1, a test sample that may contain a targetmolecule of interest (hereinafter called a ligand (1)), such as EGFR, isincubated with an aptamer construct (2) that includes an aptamer (3) anda first binding pair member (4) (e.g. biotin), that has specificaffinity for the ligand. If the sample contains the ligand, the aptamerbinds to the ligand, forming an aptamer-ligand complex (5). The aptamerdoes not bind non-target molecules in the sample with the same highaffinity and specificity as the ligand.

Referring to step B of FIG. 1, the incubated mixture is contacted with afirst solid support (10) on which is immobilized a second binding pairmember (14) that specifically binds to the first binding pair member (4)(e.g. streptavidin or an antibody that binds biotin). The aptamer-ligandcomplex (5) and uncomplexed aptamer (6) are captured on the solidsupport (10) via the second binding pair member (14). Alternatively, theaptamer complex may be captured on the solid support (10) via thebinding pair members prior to exposure to the ligand-containing sample.Supporting either event, the solid support is washed to remove unboundsample and reagent components.

Referring to step C of FIG. 1, the captured ligand (1) of theaptamer-ligand complex (5) is labeled with a third binding pair member(7) (e.g. biotin).

Referring to step D of FIG. 1, the aptamer-ligand complex (5) anduncomplexed aptamer (6) are released from the first solid support usinga method appropriate to the particular binding pair member beingemployed. For example, biotin may be attached to the aptamer using aphotocleavable linkage. When the complex is exposed to ultraviolet (UV)light, the linkage is cleaved, freeing the aptamer-ligand complex (5)and uncomplexed aptamer (6) from the first solid support. If the firstbinding pair member is not removed from the aptamer during release fromthe solid support, the first binding pair member may be optionallyremoved from the aptamer after release in order that the same bindingpair member species may be used during later processing withoutinterference.

Optionally, a kinetic challenge can be performed to increase the assayspecificity and decrease the background signal. The application of akinetic challenge to an assay employing aptamers eliminates the need toenhance specificity by introducing a second capture reagent, such as anantibody used in a sandwich ELISA assay. If a kinetic challenge isintroduced, non-specific complexes between the aptamer and anynon-target molecules are unlikely to re-form following theirdissociation. Since non-specific complexes generally dissociate morerapidly than an aptamer affinity complex, a kinetic challenge reducesthe likelihood that an aptamer will be involved in a non-specificcomplex with a non-target molecule.

Kinetic challenge molecules include any molecule that can preventnon-specific binding of a non-target molecule to an aptamer. Kineticchallenge molecules include oligonucleotides, polyanions (e.g., heparin,herring sperm DNA, single-stranded salmon sperm DNA, and polydextrans,such as dextran sulfate), abasic phosphodiester polymers, dNTPs, andpyrophosphate. A kinetic challenge molecule can also be any moleculethat can form a non-specific complex with a free aptamer, for example toprevent that aptamer from rebinding non-specifically to a non-targetmolecule. Such kinetic challenge molecules include polycations (e.g.,spermine, spermidine, polylysine, and polyarginine) and amino acids(e.g., arginine and lysine). When a competitor is used as the kineticchallenge a fairly high concentration is utilized relative to theanticipated concentration of total protein or total aptamer present inthe sample. In one embodiment, about 10 mM dextran sulfate is used asthe competitor in a kinetic challenge.

Referring to step E of FIG. 1, following release of the aptamer-ligandcomplex (5) and the uncomplexed aptamer (6) from the first solid supportand optional kinetic challenge, a fourth binding pair member (17) (e.g.streptavidin or an antibody against biotin) bound to a second solidsupport (11) is used to immobilize the released aptamer-ligand complex(5). The second solid support is then washed to remove the uncomplexedaptamers (6) and other molecules not attached to the second solidsupport.

Referring to step F of FIG. 1, following capture of aptamer-ligandcomplex on the second solid support (11), the aptamer (3) is releasedfrom the complex. Aptamer release may be affected by contacting theimmobilized complex with a solution (e.g. high pH solution) that permitsthe aptamer (3) to dissociate from the ligand (1).

Following dissociation of the aptamer (3) from the ligand (1), theamount of ligand (3) may be detected or quantitated either directly orindirectly. Ligand (1) may be measured directly by contacting the ligand(1) with a detectably labeled binding agent (e.g. an aptamer orantibody). Alternatively, or in addition, ligand (1) may be measuredindirectly by detecting the presence and/or amount of the releasedaptamer (3). Aptamers which include an oligonucleotide portion may bedetected and/or quantitated using any suitable nucleic acid detectiontechnique, such as, for example, probe hybridization (e.g. using probesimmobilized on solid substrates such as beads or a microarray, or usingdetectably-labeled probes), quantitative PCR (qPCR), mass spectroscopy(MS), a combination probe-bead PCR amplification (such as a Luminex™assay), the Invader assay, and the like. Depending on which technique isemployed, the aptamer may be designed or modified to include adetectable label if direct detection of the aptamer is desired. Aptamerlabeling may be accomplished during synthesis (either enzymatically orchemically) or at any time during the assay (i.e., at any time prior todetection).

Single-Support Sandwich Detection of EGFR

Aptamers may substitute for antibodies or other ligands that bind withspecificity and are used to detect other molecules. Thus, an analyte ina sample, e.g. free EGFR, may be detected using an aptamer specific forthe analyte in a single-support sandwich-style assay similar to anenzyme-linked immunosorbent assay (ELISA). The aptamer can be used toeither capture the analyte or to detect the analyte.

An aptamer (2) specific for a ligand (1), such as EGFR, can be used tocapture the ligand on a solid support. For example, steps A-B of FIG. 1illustrate an example of attaching an aptamer (2) to a solid support(10) and recognizing ligand, e.g. free EGFR, in a sample. Once theligand is captured by the aptamer, the ligand in the aptamer-ligandcomplex (5) is incubated with a detectably labeled binding agent (notshown in FIG. 1) to detect the ligand. Binding agents for detection caninclude detectably labeled aptamers or antibodies that recognize anyepitope of the aptamer-bound free EGFR.

Alternatively, the aptamer-bound ligand (2) can be labeled with abinding pair member (7), e.g. biotin, as illustrated in part C of FIG.1, and the binding pair member can be detected with a detectably labeledbinding agent, e.g. streptavidin or an antibody that recognizes biotin.

Aptamers can also be used to detect analytes in a single-supportsandwich assay. For example, referring to step B of FIG. 1, a bindingpair member (14), e.g. an antibody, that recognizes only free EGFR in asample can be attached to a solid support (10) and used to capture EGFRin a sample. A detectably labeled aptamer that recognizes only free EGFRcan then be used to detect only the free EGFR in the sample (e.g.aptamer labeled with Alexa Fluor 488-7-OBEA-dCTP; Life Technologies Cat.No. C21555).

Aptamer Modification

Nucleotides in aptamers can be chemically modified, singly or in anycombination. Nucleotide chemical modifications in aptamers may include,for example, 2′-position sugar modifications, 5-position pyrimidinemodifications (e.g., 5-(N-benzylcarboxyamide)-2′-deoxyuridine,5-(N-isobutylcarboxyamide)-2′-deoxyuridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-naphthylmethylcarboxamide)-2′-deoxyuridine, or5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine),modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo- or 5-iodo-uracil, backbone modifications,methylations, unusual base-pairing combinations such as the isobases,isocytidine and isoguanidine, and the like.

In some embodiments, the term “C-5 modified pyrimidine” refers to apyrimidine with a modification at the C-5 position including, but notlimited to, those moieties shown in FIG. 5. Examples of a C-5 modifiedpyrimidine include those described in U.S. Pat. Nos. 5,719,273 and5,945,527. Examples of a C-5 modification include substitution ofdeoxyuridine at the C-5 position with a substituent selected from:benzylcarboxyamide (alternatively benzylaminocarbonyl) (Bn),naphthylmethylcarboxyamide (alternatively naphthylmethylaminocarbonyl)(Nap), tryptaminocarboxyamide (alternatively tryptaminocarbonyl) (Trp),isobutylcarboxyamide (alternatively isobutylaminocarbonyl) (iBu),tyrosylcarboxyamide (alternatively tyrosylaminocarbonyl) (Tyr),2-naphthylmethylcarboxyamide (alternatively2-naphthylmethylaminocarbonyl) (2Nap) and phenethyl-1-carboxyamide(alternatively phenethyl-1-aminocarbonyl) (PE), as illustratedimmediately below.

Aptamer modifications may include nucleotide additions or modificationsat the 3′ and 5′ ends, such as a cap or pegylation. Other modificationscan include substitution of one or more of the naturally occurringnucleotides with an analog, internucleotide modifications such as, forexample, those with uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, etc.) and those withcharged linkages (e.g., phosphorothioates, phosphorodithioates, etc.),those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, and those with modifiedlinkages (e.g., alpha anomeric nucleic acids, etc.). Further, any of thehydroxyl groups ordinarily present in a sugar may be replaced by aphosphonate group or a phosphate group; protected by standard protectinggroups; or activated to prepare additional linkages to additionalnucleotides or to a solid support. The 5′ and 3′ terminal OH groups canbe phosphorylated or substituted with amines, organic capping groupmoieties of from about 1 to about 20 carbon atoms, or organic cappinggroup moieties of from about 1 to about 20 polyethylene glycol (PEG)polymers or other hydrophilic or hydrophobic biological or syntheticpolymers. If present, a modification to the nucleotide structure may beimparted before or after assembly of a polymer. A sequence ofnucleotides may be interrupted by non-nucleotide components. Apolynucleotide may be further modified after polymerization, such as byconjugation with a labeling component.

Inverted nucleotides may be used as cap on the 3′ or 5′ end of a nucleicacid. As used herein, the term “inverted nucleotide” meansdeoxyribonucleotides or ribonucleotides having an orientation oppositeto that of the sequence that contains them. Classically, with synthesistaking place in the 3′ to 5′ direction, inverted nucleotides areintroduced following or before the rest of the nucleotide strand, in the5′ to 3′ direction. The inverted nucleotides and the rest of the strandare then joined together by a 5′-5′ or 3′-3′ bond. Reference may be madeto Koga M., et al., J. Org. Chem., 1991, 56, 3757. The invertednucleotides can optionally include at least one modified nucleotide, forexample at least one nucleotide having a modified nucleic acid base,such as inosine, methyl-5-deoxycytidine, dimethylamino-5-deoxyuridine,deoxyuridine, diamino-2,6-purine, bromo-5-deoxyuridine or any othermodified base permitting hybridization.

Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclicsugar analogs, α-anomeric sugars, epimeric sugars such as arabinose,xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses,acyclic analogs and abasic nucleoside analogs such as methyl riboside.As noted above, one or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups includeembodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S(“dithioate”), (O)NR₂ (“amidate”), P(O)R′, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. Substitution ofanalogous forms of sugars, purines, and pyrimidines can be advantageousin designing a final product, as can alternative backbone structureslike a polyamide backbone, for example.

In some embodiments, an aptamer has the nucleic acid sequence of SEQ IDNO: 1, in which each “N” is, independently, a 5-position modified2′-deoxyuridine including but not limited to, for example,5-benzylaminocarbonyl-2′-deoxyuridine,5-(N-benzylcarboxyamide)-2′-deoxyuridine,5-(N-isobutylcarboxyamide)-2′-deoxyuridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-naphthylmethylcarboxamide)-2′-deoxyuridine, or5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine.

(SEQ ID NO: 1) ANCGAGGNNGNGGGNCGGANNGNNGGANNCNNNAAGNNGGGAACACCAACCGAGAACG 

In some embodiments, an aptamer includes the nucleic acid sequence ofSEQ ID NO: 2, in which each “N” is, independently, a 5-position modified2′-deoxyuridine including but not limited to, for example,5-benzylaminocarbonyl-2′-deoxyuridine,5-(N-benzylcarboxyamide)-2′-deoxyuridine,5-(N-isobutylcarboxyamide)-2′-deoxyuridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-naphthylmethylcarboxamide)-2′-deoxyuridine, or5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine.

(SEQ ID NO: 2) NNGNNGGANNCNNNAAG 

In some embodiments, an aptamer includes the nucleic acid sequence ofSEQ ID NO: 5, in which each “N” is, independently, a 5-position modified2′-deoxyuridine including but not limited to, for example,5-benzylaminocarbonyl-2′-deoxyuridine,5-(N-benzylcarboxyamide)-2′-deoxyuridine,5-(N-isobutylcarboxyamide)-2′-deoxyuridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-naphthylmethylcarboxamide)-2′-deoxyuridine, or5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine.

(SEQ ID NO: 5) ANCGAGGNNGNGGGNCGGANNGNNGGANNCNNNAAGNNGGGAACACCAAC

In some embodiments, an aptamer includes the nucleic acid sequence ofSEQ ID NO: 6, in which each “N” is, independently, a 5-position modified2′-deoxyuridine including but not limited to, for example,5-benzylaminocarbonyl-2′-deoxyuridine,5-(N-benzylcarboxyamide)-2′-deoxyuridine,5-(N-isobutylcarboxyamide)-2′-deoxyuridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-naphthylmethylcarboxamide)-2′-deoxyuridine, or5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine.

(SEQ ID NO: 6) ANCGAGGNNGNGGGNCGGANNGNNGGANNCNNNAAGNNGG 

In some embodiments, an aptamer includes the nucleic acid sequence ofSEQ ID NO: 7, in which each “N” is, independently, a 5-position modified2′-deoxyuridine including but not limited to, for example,5-benzylaminocarbonyl-2′-deoxyuridine,5-(N-benzylcarboxyamide)-2′-deoxyuridine,5-(N-isobutylcarboxyamide)-2′-deoxyuridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-naphthylmethylcarboxamide)-2′-deoxyuridine, or5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine.

(SEQ ID NO: 7) GAGNNGNANGGGGNCGGANNGNNGGANNCNNNAAGNNCGG 

In some embodiments, an aptamer includes the nucleic acid sequence ofSEQ ID NO: 8, in which each “N” is, independently, a 5-position modified2′-deoxyuridine including but not limited to, for example,5-benzylaminocarbonyl-2′-deoxyuridine,5-(N-benzylcarboxyamide)-2′-deoxyuridine,5-(N-isobutylcarboxyamide)-2′-deoxyuridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-naphthylmethylcarboxamide)-2′-deoxyuridine, or5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine.

(SEQ ID NO: 8) GGGNCGGANNGNNGGANNCNNNAAGNN 

In some embodiments, the present disclosure provides an aptamer thatspecifically binds to EGFR and includes a primary nucleic acid sequence.In one embodiment, the primary nucleic acid sequence is selected fromSEQ ID NOS: 1 to 8. In other embodiments, the primary nucleic acidsequence is selected such that it is at least 75% identical, at leastabout 80% identical, at least about 85% identical, at least about 90%percent identical, at least about 91% identical, at least about 92%identical, at least about 93% identical, at least about 94% identical,or at least about 95% identical to a primary nucleic acid sequenceselected from SEQ ID NOS: 1 to 8.

The terms “sequence identity”, “percent sequence identity”, “percentidentity”, “% identical”, “% identity”, and variations thereof, whenused in the context of two nucleic acid sequences, are usedinterchangeably to refer to the number of nucleotide bases that are thesame in a query nucleic acid or a portion of a query nucleic acid, whenit is compared and aligned for maximum correspondence to a referencenucleic acid, divided by either (1) the number of nucleotide bases inthe query sequence between and including the most 5′ corresponding(i.e., aligned) nucleotide base and the most 3′ corresponding (i.e.,aligned) nucleotide base, or (2) the total length of the referencesequence, whichever is greater. Exemplary alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith and Waterman, Adv. Appl. Math., 2:482, 1981, by the homologyalignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443,1970, by the search for similarity method of Pearson and Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by visual inspection (see generally, Ausubel, F. M.,et al., Current Protocols in Molecular Biology, pub. by GreenePublishing Assoc. and Wiley-Interscience (1987)).

One example of an algorithm that is suitable for determining percentsequence identity is the algorithm used in the basic local alignmentsearch tool (hereinafter “BLAST”), see, e.g. Altschul, et al., J. Mol.Biol. 215:403-410, 1990 and Altschul, et al., Nucleic Acids Res.,15:3389-3402, 1997. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(hereinafter “NCBI”). The default parameters used in determiningsequence identity using the software available from NCBI, e.g., BLASTN(for nucleotide sequences) are described in McGinnis, et al., NucleicAcids Res., 32:W20-W25, 2004.

As used herein, when describing the percent identity of a nucleic acid,such as a EGFR aptamer, the sequence of which is at least, for example,about 95% identical to a reference nucleotide sequence, it is intendedthat the nucleic acid sequence is identical to the reference sequenceexcept that the nucleic acid sequence may include up to five pointmutations per each 100 nucleotides of the reference nucleic acidsequence. In other words, to obtain a desired nucleic acid sequence, thesequence of which is at least about 95% identical to a reference nucleicacid sequence, up to 5% of the nucleotides in the reference sequence maybe deleted or substituted with another nucleotide, or some number ofnucleotides up to 5% of the total number of nucleotides in the referencesequence may be inserted into the reference sequence (referred to hereinas an insertion). These mutations of the reference sequence to generatethe desired sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Further, it is intended that a nucleotide base isconsidered “identical” for the purposes of determining percent identity,when the nucleotide base (1) is the same as the nucleotide base in thereference sequence, or (2) is derived from the nucleotide base in thereference sequence, or (3) is derived from the same nucleotide base fromwhich the nucleotide base in the reference sequence is derived. Forexample, 5-methyl cytosine is considered to be “identical” to cytosinefor the purposes of calculating percent identity. Similarly, themodified uridines shown in FIG. 5 are considered to be identical to oneanother for the purpose of determining percent identity. The referencesequence may be any one of the entire nucleotide sequences shown in SEQID NOS: 1 to 8, or any fragment of any of these sequences.

Aptamer Labeling

Aptamers may be labeled with a detectable label using several methodsknown by those having ordinary skill in the art. Signals from thedetectable label may be detected by various means and will depend on thenature of the detectable label. Detectable labels include but are notlimited to fluorophores, isotopes (e.g., ³²P, ³³P, ³⁵S, ³H, ¹⁴C, ¹²⁵,¹³¹I), electron-dense reagents (e.g., gold, silver), nanoparticles,enzymes commonly used in an ELISA (e.g., horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescentcompound, colorimetric labels (e.g., colloidal gold), magnetic labels(e.g., Dynabeads™), biotin, digoxigenin, haptens, proteins for whichantisera or monoclonal antibodies are available, ligands, hormones,oligonucleotides capable of forming a complex with the correspondingoligonucleotide complement.

Detectable labels may be incorporated into nucleic acids by covalent ornon-covalent means, e.g., by transcription, such as by random-primerlabeling using Klenow polymerase, or nick translation, or,amplification, or equivalent as is known in the art. For example, anucleotide base is conjugated to a detectable moiety, such as afluorescent dye, e.g., Cy3™ or Cy5™ and then incorporated into nucleicacid probes during nucleic acid synthesis or amplification. Nucleic acidprobes may be labeled when synthesized using Cy3™- or Cy5™-dCTPconjugates mixed with unlabeled dCTP.

For example, fluorescently-labeled nucleotides are commerciallyavailable and may be enzymatically incorporated into oligonucleotides(e.g. ChromaTide™ nucleotides from Life Technologies/Invitrogen,described in The Molecular Probes Handbook, 11^(th) Edition, section8.2). In another exemplary method, amine-reactive nucleotides may beincorporated into an oligonucleotide and then amine-reacted with adesired fluorophore (e.g. ARES™ Nucleic Acid Labeling Kits from LifeTechnologies/Invitrogen, described in The Molecular Probes Handbook,11^(th) Edition, section 8.2). Another method of labelingoligonucleotides utilizes a platinum-based chemistry system to linkmolecules with guanine-based nucleotides (e.g. ULYSIS™ Nucleic AcidLabeling Kits from Life Technologies/Invitrogen, described in TheMolecular Probes Handbook, 11^(th) Edition, section 8.2).

Nucleic acid probes may be labeled by non-covalent means known in theart. For example, Kreatech Biotechnology's Universal Linkage System®(ULS®) provides a non-enzymatic labeling technology, wherein a platinumgroup forms a co-ordinative bond with DNA, RNA or nucleotides by bindingto the N7 position of guanosine. This technology may also be used tolabel proteins by binding to nitrogen and sulfur containing side chainsof amino acids. See, e.g., U.S. Pat. Nos. 5,580,990; 5,714,327; and5,985,566; and European Patent No. 0539466.

Labeling with a detectable label also may include nucleic acid probesattached to another biological molecule, such as a nucleic acid, e.g.,an oligonucleotide, or a nucleic acid in the form of a stem-loopstructure as a “molecular beacon” or an “aptamer beacon”. Molecularbeacons as detectable moieties are well known in the art; for example,Sokol (Proc. Natl. Acad. Sci. USA (1998), 95:11538-11543) synthesized“molecular beacon” reporter oligodeoxynucleotides with matchedfluorescent donor and acceptor chromophores on their 5′ and 3′ ends. Inthe absence of a complementary nucleic acid strand, the molecular beaconremains in a stem-loop conformation where fluorescence resonance energytransfer prevents signal emission. On hybridization with a complementarysequence, the stem-loop structure opens increasing the physical distancebetween the donor and acceptor moieties thereby reducing fluorescenceresonance energy transfer and allowing a detectable signal to be emittedwhen the beacon is excited by light of the appropriate wavelength. Seealso, e.g., Antony (Biochemistry (2001), 40:9387-9395,), describing amolecular beacon consist of a G-rich 18-mer triplex formingoligodeoxyribonucleotide. See also U.S. Pat. Nos. 6,277,581 and6,235,504.

Aptamer beacons are similar to molecular beacons; see, e.g., Hamaguchi,Anal. Biochem. (2001), 294:126-131; Poddar, Mol. Cell. Probes (2001),15:161-167; Kaboev, Nucleic Acids Res. (2000), 28:E94. Aptamer beaconsmay adopt two or more conformations, one of which allows ligand binding.A fluorescence-quenching pair is used to report changes in conformationinduced by ligand binding. See also, e.g., Yamamoto, et al., Genes Cells(2000), 5:389-396; Smimov, et al., Biochemistry (2000), 39:1462-1468.

The nucleic acid probe may be indirectly detectably labeled via apeptide. A peptide can be made detectable by incorporating predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies,transcriptional activator polypeptide, metal binding domains, epitopetags). A label may also be attached via a second peptide that interactswith the first peptide (e.g., S-S association).

Solid Supports

A solid support may take any of a variety of configurations ranging fromsimple to complex. The solid support can have anyone of a number ofshapes, including a strip, plate, disk, rod, particle, bead, tube, well(microtiter), and the like. The solid support may be porous ornon-porous, magnetic, paramagnetic, or non-magnetic, polydisperse ormonodisperse, hydrophilic or hydrophobic. The solid support may also bein the form of a gel or slurry of closely-packed (as in a column matrix)or loosely-packed particles.

The material of a solid support is generally capable of enduringconditions related to the attachment of the binding pair members orprobes to the surface and any subsequent treatment, handling, orprocessing encountered during the performance of an assay. The materialsmay be naturally occurring, synthetic, or a modification of a naturallyoccurring material. Suitable solid support materials may includesilicon, a silicon wafer chip, graphite, mirrored surfaces, laminates,membranes, ceramics, plastics (including polymers such as, e.g., latex,poly(vinyl chloride), cyclo-olefin copolymers, agarose gels or beads,polyacrylamide, polyacrylate, polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), polytetrafluoroethylene (PTFE or Teflon®), nylon,poly(vinyl butyrate)), germanium, gallium arsenide, gold, silver,Langmuir Blodgett films, a flow through chip, etc., either used bythemselves or in conjunction with other materials. Additional rigidmaterials may be considered, such as glass, which includes silica andfurther includes, for example, glass that is available as Bioglass.Other materials that may be employed include porous materials, such as,for example, controlled pore glass beads, crosslinked beaded Sepharose®or agarose resins, or copolymers of crosslinked bis-acrylamide andazalactone. Other beads include polymer beads, solid core beads,paramagnetic beads, or microbeads. Any other materials known in the artthat are capable of having one or more functional groups, such as any ofan amino, carboxyl, thiol, or hydroxyl functional group, for example,incorporated on its surface, are also contemplated.

Non-Covalent Attachment of Aptamers to a Solid Support

Aptamer constructs can be attached to a solid support non-covalentlyusing one binding pair member attached to a solid support (covalently ornon-covalently) and one binding pair member attached to the aptamer.Binding pairs include biotin and streptavidin, antibody and antigen, theFab region of an antibody and its antigen, protein A and the Fc domainof IgG, and a single oligonucleotide strand and its complement.

In some embodiments, the binding pair for attaching the aptamer isbiotin and streptavidin, wherein the aptamer is biotinylated and thesolid support is coated with streptavidin. Methods for biotinylatingnucleic acid are known in the art (e.g. by photo-cross linking usingEZ-link psoralen-PEO biotin from Pierce Chemical Co., by chemicalcoupling using Label IT® μArray® Biotin Labeling Kit from Minis BioCorp., PFP Biotin from Pierce Chemical Co., by nick translation usingBioNick DNA Labeling System from Invitrogen corporation, or by 3′-endlabeling using commercially available kits e.g. Biotin 3-end labelingkit from Pierce).

In other embodiments, the binding pair consists of a ligand-receptor, ahormone-receptor, and an antigen-antibody. Examples of such binding pairinclude but are not limited to digoxigenin and anti-digoxigeninantibody; 6-(2,4-dinitrophenyl)aminohexanoic acid and anti-dinitrophenylantibody; 5-Bromo-dUTP (BrdUTP) and anti-BrdUTP antibody; N-acetyl2-aminofluorene (AAF) and anti-AAF antibody. The solid surfaces in thesecases consist of the antibody, and the genomic nucleic acid is modifiedto consist of the antigen. Methods of incorporating digoxigenin,2,4-dinitrophenyl group, 5-Bromo-dUTP group into DNA can be achieved bynick translation, or by terminal transferase reaction, examples of whichare amply documented in the art or may be achieved by using commerciallyavailable kits e.g. kits DIG DNA labeling kit from Roche AppliedSciences. Digoxigenin can be chemically coupled to the nucleic acid withDigoxigenin-NHS-ester. N-acetyl 2-aminofluorene (AAF) can be covalentlycoupled to the genomic nucleic acid.

Covalent Attachment of Aptamers to a Solid Support

Aptamer can also be covalently attached to a solid support usingfunctionalization chemistry for creating microarrays ornucleotide-coated beads. If covalent bonding between the genomic nucleicacid and the surface is desired, the solid surface will usually befunctional or be capable of being functionalized. Examples of functionalgroups used for linking include but are not limited to carboxylic acids,aldehydes, amino groups, cyano groups, ethylenic groups, hydroxylgroups, thiol groups.

For example, methods for synthesizing oligonucleotides from nucleicacids attached to solid supports have been described (see, e.g., U.S.Pat. No. 5,436,327, U.S. Pat. No. 5,800,992, U.S. Pat. No. 5,445,934,U.S. Pat. No. 5,763,170, U.S. Pat. No. 5,599,695 and U.S. Pat. No.5,837,832). Solid supports can also be thiol- or amine-functionalizedand then reacted with an oligonucleotide having the properly modifiednucleic acid.

In some embodiments, the solid support may be coated with epoxy group,amino group, mercapto group, or polylysine. Coated solid supports areavailable commercially e.g. beads coated with functional groups areavailable from Invitrogen Corporation, BD Biosciences; glass slidescoated with functional groups are available from Pierce, Asper Biotech,Full Moon Biosystems, and ThermoFisher Inc.

The aptamer may be modified to consist of functional groups. The 5′phosphate group of genomic nucleic acid, may be conjugated to primaryamine-containing molecules using the carbodiimide crosslinker EDC(Pierce Product No. 22980) and imidazole. The 5′ phosphate group of anucleic acid may be modified to consist of amine group with an excess ofethylenediamine, and using carbodiimide crosslinker EDC (Pierce ProductNo. 22980) and imidazole as described in Pierce Technote No. 30.Depending on the amine containing molecules used, the crosslinkingstrategy can be adapted in a number of ways to directly or indirectlymodify, label or conjugate genomic nucleic acid. For example, to createa photoactivable (random-reactive) nucleic acid, p-azidobenzoylhydrazide, (ABH, Pierce catalog No. 21510) may be used instead ofethylenediamine in the default reaction. To create a sulfhydryl-reactivenucleic acid, [N-e-Maleimidocaproic acid]hydrazide, trifluoroacetic acidsalt (EMCH, Pierce catalog No. 22106), N-[k-Maleimidoundecanoicacid]hydrazide, (KMUH Pierce catalog No. 22111), or4-(4-N-Maleimidophenyl)butyric acid hydrazide hydrochloride, (MPBHPierce catalog No. 22305) may be used instead of ethylenediamine in thedefault reaction. To obtain a sulfhydryl crosslink that is reversible3-(2-Pyridyldithio)propionyl hydrazide (PDPH, Pierce catalog No. 22301)may be used instead of ethylenediamine. This strategy is useful forlinking genomic nucleic acid to sulfhydryl-containing solid support. Tocreate a sulfhydryl group on genomic nucleic acid cystamine(NH₂—CH₂—CH₂—S—S—CH₂—CH₂—NH₂) may be used instead of ethylenediamine inthe default reaction, and then reduce the disulfide bond with DTT orsimilar reagent. This strategy is useful for covalently coupling tomaleimide activated solid support. To immobilize nucleic acid to abeaded affinity support, UltraLink Hydrazide (Pierce catalog No. 53149)may be used instead of ethylenediamine in the default reaction.

The manner of linking a wide variety of functional groups to each otheris well known and is amply illustrated in the literature. In oneembodiment, the chemical linkers may be used to covalently link twofunctional groups, one on the solid support and the other on the genomicnucleic acid. The chemical linkers may be mono functional, bifunctional,polyfunctional, hetero-bifunctional, or hetero-polyfunctional. In someembodiments, the chemical linkers may have spacer arms to avoid sterichindrance. Examples of chemical linkers to couple amino group to anamino group include but are not limited to ethylene glycolbis[succinimidylsuccinate], disuccinimidyl suberate,1,5-difluoro-2,4-dinitrobenzene. Examples of chemical linkers to couplethiol group to a thiol group include but are not limited to1,4-di-[3′-(2′-pyridyldithio)-propionamido]butane,dithio-bismaleimidoethane. A wide variety of suitable cross linkers andthe methods of cross linking are available from Pierce.

In another embodiment, the genomic nucleic acid is anchored to the solidsupport through photoactive moieties. In one embodiment, the solidsurface may anchor photoactive moieties capable of coupling the genomicnucleic acid by photo activation. In another embodiment, the 5′phosphate group of genomic nucleic acid may be conjugated to aphotoactive group, capable of photocrosslinking to functional groups onthe solid surface. In another embodiment, the genomic nucleic acid maybe anchored to the solid surface through a linker having two or morephotoactive moieties, one or more at each end, wherein the linkercouples to the solid surface and to the genomic nucleic acid uponexposing the solid surface and genomic nucleic acid in presence of thelinker with radiation of suitable wavelength. Examples of photoactivemoieties include but not limited to azides, aryl azides, azidophenacyl,4-nitrophenyl 3-diazopyruvate, psolarens, psolaren derivatives.

In another embodiment, the aptamer can be cross-linked to nylon,nitrocellulose, or nylon-reinforced nitrocellulose membranes, byexposing the solid surface and the nucleic acid to ultra-violetradiation. The manner of cross-linking linking of nucleic acid tovarious surfaces is well known and is amply illustrated in theliterature (e.g. using Stratagene UV crosslinker).

Aptamer Amplification

Nucleic acid aptamers may be amplified by various methods known to theskilled artisan. Nucleic acid amplification may be linear orexponential. Amplification is generally carried out using polymerasechain reaction (PCR) technologies known in the art. See e.g., Mullis andFaloona, Methods Enzymol. (1987), 155:335, U.S. Pat. Nos. 4,683,202,4,683,195 and 4,800,159.

Oligonucleotide tags may be used to detect and identify aptamers. Forexample, a known unique sequence of nucleic acids can be added to the 5′or 3′ end of an aptamer that can be identified using PCR techniquesknown in the art. The tag may be recognized by primers used to amplifythe aptamer

Alternative methods to PCR include for example, isothermal amplificationmethods, rolling circle methods, Hot-start PCR, real-time PCR,Allele-specific PCR, Assembly PCR or Polymerase Cycling Assembly (PCA),Asymmetric PCR, Colony PCR, Emulsion PCR, Fast PCR, Real-Time PCR,nucleic acid ligation, Gap Ligation Chain Reaction (Gap LCR),Ligation-mediated PCR, Multiplex Ligation-dependent Probe Amplification,(MLPA), Gap Extension Ligation PCR (GEXL-PCR), quantitative PCR (Q-PCR),Quantitative real-time PCR (QRT-PCR), multiplex PCR, Helicase-dependentamplification, Intersequence-specific (ISSR) PCR, Inverse PCR,Linear-After-The-Exponential-PCR (LATE-PCR), Methylation-specific PCR(MSP), Nested PCR, Overlap-extension PCR, PAN-AC assay, ReverseTranscription PCR (RT-PCR), Rapid Amplification of cDNA Ends (RACE PCR),Single molecule amplification PCR (SMA PCR), Thermal asymmetricinterlaced PCR (TAIL-PCR), Touchdown PCR, long PCR, nucleic acidsequencing (including DNA sequencing and RNA sequencing), transcription,reverse transcription, duplication, DNA or RNA ligation, and othernucleic acid extension reactions known in the art. The skilled artisanwill understand that other methods may be used either in place of, ortogether with, PCR methods, including enzymatic replication reactionsdeveloped in the future. See, e.g., Saiki, “Amplification of GenomicDNA” in PCR Protocols, Innis, et al., eds., Academic Press, San Diego,Calif., 13-20 (1990); Wharam, et al., 29(11) Nucleic Acids Res, E54-E54(2001); Hafner, et al., 30(4) Biotechniques, 852-6, 858, 860 passim(2001).

Detection of Amplified Aptamer

Amplification of nucleic acid aptamers can be detected by any of anumber of methods well-known in the art such as gel electrophoresis,column chromatography, hybridization with a probe, or sequencing.

One general method for real time PCR uses fluorescent probes such as theTaqMan® probes (Heid, et al., Genome Res 6: 986-994, 1996), molecularbeacons, and Scorpions™. Real-time PCR quantifies the initial amount ofthe template with more specificity, sensitivity and reproducibility,than other forms of quantitative PCR, which detect the amount of finalamplified product. Real-time PCR does not detect the size of theamplicon. The probes employed in Scorpion™ and TaqMan® technologies arebased on the principle of fluorescence quenching and involve a donorfluorophore and a quenching moiety.

The term “donor fluorophore” as used herein means a fluorophore that,when in close proximity to a quencher moiety, donates or transfersemission energy to the quencher. As a result of donating energy to thequencher moiety, the donor fluorophore will itself emit less light at aparticular emission frequency that it would have in the absence of aclosely positioned quencher moiety.

The term “quencher moiety” as used herein means a molecule that, inclose proximity to a donor fluorophore, takes up emission energygenerated by the donor and either dissipates the energy as heat or emitslight of a longer wavelength than the emission wavelength of the donor.In the latter case, the quencher is considered to be an acceptorfluorophore. The quenching moiety can act via proximal (i.e.,collisional) quenching or by Förster or fluorescence resonance energytransfer (“FRET”). Quenching by FRET is generally used in TaqMan® probeswhile proximal quenching is used in molecular beacon and Scorpion™ typeprobes.

Suitable fluorescent moieties include but are not limited to thefollowing fluorophores working individually or in combination:

4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives: acridine, acridine isothiocyanate; Alexa Fluors: AlexaFluor® 350, Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor® 555, AlexaFluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (Molecular Probes);5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS); N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BlackHole Quencher™ (BHQ™) dyes (biosearch Technologies); BODIPY dyes:BODIPY® R-6G, BOPIPY® 530/550, BODIPY® FL; Brilliant Yellow; coumarinand derivatives: coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumarin 151); Cy2®, Cy3®, Cy3.5®,Cy5®, Cy5.5®; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); Eclipse™(Epoch Biosciences Inc.); eosin and derivatives: eosin, eosinisothiocyanate; erythrosin and derivatives: erythrosin B, erythrosinisothiocyanate; ethidium; fluorescein and derivatives:5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein(DTAF), 2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE),fluorescein, fluorescein isothiocyanate (FITC),hexachloro-6-carboxyfluorescein (HEX), QFITC (XRITC),tetrachlorofluorescein (TET); fluorescamine; IR144; IR1446; lanthamidephosphors; Malachite Green isothiocyanate; 4-methylumbelliferone; orthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin, R-phycoerythrin; allophycocyanin; o-phthaldialdehyde;Oregon Green®; propidium iodide; pyrene and derivatives: pyrene, pyrenebutyrate, succinimidyl 1-pyrene butyrate; QSY® 7; QSY® 9; QSY® 21; QSY®35 (Molecular Probes); Reactive Red 4 (Cibacron® Brilliant Red 3B-A);rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride,rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine green, rhodamineX isothiocyanate, riboflavin, rosolic acid, sulforhodamine B,sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101(Texas Red); terbium chelate derivatives;N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC).

Molecular beacons as detectable moieties are well known in the art; forexample, Sokol (Proc. Natl. Acad. Sci. USA (1998), 95:11538-11543)synthesized “molecular beacon” reporter oligodeoxynucleotides withmatched fluorescent donor and acceptor chromophores on their 5′ and 3′ends. In the absence of a complementary nucleic acid strand, themolecular beacon remains in a stem-loop conformation where fluorescenceresonance energy transfer prevents signal emission. On hybridizationwith a complementary sequence, the stem-loop structure opens increasingthe physical distance between the donor and acceptor moieties therebyreducing fluorescence resonance energy transfer and allowing adetectable signal to be emitted when the beacon is excited by light ofthe appropriate wavelength. See also, e.g., Antony (Biochemistry (2001),40:9387-9395,), describing a molecular beacon consist of a G-rich 18-mertriplex forming oligodeoxyribonucleotide. See also U.S. Pat. Nos.6,277,581 and 6,235,504.

Aptamer beacons are similar to molecular beacons; see, e.g., Hamaguchi,Anal. Biochem. (2001), 294:126-131; Poddar, Mol. Cell. Probes (2001),15:161-167; Kaboev, Nucleic Acids Res. (2000), 28:E94. Aptamer beaconscan adopt two or more conformations, one of which allows ligand binding.A fluorescence-quenching pair is used to report changes in conformationinduced by ligand binding. See also, e.g., Yamamoto, et al., Genes Cells(2000), 5:389-396; Smimov, et al., Biochemistry (2000), 39:1462-1468.

Detection of Nucleic Acid Aptamer by Size

Methods for detecting the presence or amount of nucleic acid are wellknown in the art and any of them can be used in the methods describedherein so long as they are capable of separating individual nucleic acidby the difference in size of the amplicons. The separation techniqueused should permit resolution of nucleic acid as long as they differfrom one another by at least one nucleotide. The separation can beperformed under denaturing or under non-denaturing or nativeconditions—i.e., separation can be performed on single- ordouble-stranded nucleic acids. It is preferred that the separation anddetection permits detection of length differences as small as onenucleotide. It is further preferred that the separation and detectioncan be done in a high-throughput format that permits real time orcontemporaneous determination of amplicon abundance in a plurality ofreaction aliquots taken during the cycling reaction. Useful methods forthe separation and analysis of the amplified products include, but arenot limited to, electrophoresis (e.g., agarose gel electrophoresis,capillary electrophoresis (CE)), chromatography (HPLC), and massspectrometry.

Detection of Nucleic Acid Aptamer by Sequencing

In some examples, detection of nucleic acid is by sequencing. Sequencingmay be carried out by the dideoxy chain termination method of Sanger, etal. (Proceedings of the National Academy of Sciences USA (1977), 74,5463-5467) with modifications by Zimmermann, et al. (Nucleic Acids Res.(1990), 18:1067). Sequencing by dideoxy chain termination method can beperformed using Thermo Sequenase (Amersham Pharmacia, Piscataway, N.J.),Sequenase reagents from US Biochemicals or Sequatherm sequencing kit(Epicenter Technologies, Madison, Wis.). Sequencing may also be carriedout by the “RR dRhodamine Terminator Cycle Sequencing Kit” from PEApplied Biosystems (product no. 403044, Weiterstadt, Germany), TaqDyeDeoxy™ Terminator Cycle Sequencing kit and method(Perkin-Elmer/Applied Biosystems) in two directions using an AppliedBiosystems Model 373A DNA or in the presence of dye terminators CEQ™ DyeTerminator Cycle Sequencing Kit, (Beckman 608000). Alternatively,sequencing can be performed by a method known as Pyrosequencing(Pyrosequencing, Westborough, Mass.). Detailed protocols forPyrosequencing can be found in: Alderborn, et al., Genome Res. (2000),10:1249-1265.

Detecting Free EGFR in a Patient Sample

Epithelial carcinomas are the most common type of cancer in humans, asepithelial cells are found in organs throughout the body. Carcinomas candevelop from squamous cells in organ linings (e.g. skin, lips, mouth,esophagus, bladder, prostate, lung, ovaries, uterus, vagina, cervix, orcolon), from adenomatous cells in glands (adenocarcinomas, e.g. lung,prostate, urachus, ovaries, uterus, vagina, breast, esophagus, thyroid,parathyroid, pancreas, or stomach), or from transitional cells (e.g.kidney, bladder, ovaries, prostate, uterus, cervix, ureter, urethra, orurachus). Epithelial carcinomas also occur in brain tissues such as thechoroid plexus.

The extracellular domain of EGFR is shed in both healthy people as wellas patients with ovarian, lung, bladder, prostate, breast, and coloncancer (Carney, Expert Rev Mol Diagn. 2007 May; 7(3):309-19), and serumlevels of EGFR in patients with cancer are known to vary from EGFRlevels measured in healthy controls. For example, serum EGFR levels(extracellular domain) in patients with head and neck cancer, NSCLC,breast cancer, ovarian cancer, colon cancer, bladder cancer, andprostate cancer have been shown to be lower than those in healthycontrols. In contrast, serum EGFR levels in pancreatic, thyroid,cervical, gastric and pituitary carcinomas are higher than in healthycontrols (Lemos-Gonzalez, et al., Br J Cancer. 2007 May 21;96(10):1569-78). Furthermore, some patients have cancer that isresistant or becomes resistant to treatment with cetuximab orpanitumumab (see Carney, Expert Rev Mol Diagn. 2007 May; 7(3):309-19;Wheeler, et al., Oncogene. 2008 Jun. 26; 27(28): 3944-3956; Bardelli, etal., J Clin Oncol. 2010 Mar. 1; 28(7):1254-61).

Monitoring levels of free EGFR in samples from cancer patients receivingtreatment with therapeutic molecules that bind EGFR (e.g. cetuximab orpanitumumab) can provide information about the cancer's response totreatment. By comparing the amount of free EGFR relative to bound EGFRin a patient being treated with a therapeutic EGFR-binding molecule, atreatment provider can determine whether the therapeutic EGFR-bindingmolecule is binding sufficient amounts of free EGFR to effectively treatthat particular cancer. A cancer patient being treated with atherapeutic EGFR-binding molecule can be monitored at various timepoints during the course of treatment for possible changes in the amountof free EGFR relative to bound EGFR. Such changes can indicate that thecancer is responding to the treatment or becoming resistant to thetreatment.

Identification of Consensus Sequence

A SELEX experiment was run in which approximately 1425 sequences to EGFRwere isolated. FIG. 10 sets forth the number of unique sequencesidentified including the number of copies of each sequence. Withreference to FIG. 10, it can be seen that 21 unique sequences wereisolated. FIG. 10 shows the alignment of the 21 sequences relative toeach other. FIG. 11 summarizes the consensus sequence as a frequencymatrix in (1) unique counts and (2) total copy counts. From thisinformation the following consensus sequence was identified:

(SEQ ID NO: 55) XGANNGNNYGANNCNN;wherein X is G, C, or A; Y is G or A and each “N” is, independently, a5-position modified 2′-deoxyuridine including but not limited to, forexample, 5-(N-benzylcarboxyamide)-2′-deoxyuridine,5-(N-isobutylcarboxyamide)-2′-deoxyuridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-naphthylmethylcarboxamide)-2′-deoxyuridine, or5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine. Many ofthese nucleotide modifications are anticipated to be equally effectivein promoting high affinity binding to the EGF Receptor (EGFR) andproviding a slow off rate.

Thus in one aspect, with reference to FIGS. 10 and 11 the presentdisclosure includes an aptamer to the EGF Receptor selected from:

(SEQ ID NO: 55) XGANNGNNYGANNCNN;wherein X is G, C, or A; Y is G or A and each “N” is, independently, a5-position modified 2′-deoxyuridine including but not limited to, forexample, 5-benzylaminocarbonyl-2′-deoxyuridine,5-(N-benzylcarboxyamide)-2′-deoxyuridine,5-(N-isobutylcarboxyamide)-2′-deoxyuridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-naphthylmethylcarboxamide)-2′-deoxyuridine, or5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine. In oneembodiment each “N” is a 5-(N-benzylcarboxyamido)-2′-deoxyuridine.

In some aspects, N is further modified at the 2′-position with a moietyselected from the group including, but not limited to for example2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, etc. asdescribed above.

In another embodiment, the present disclosure provides an aptamer thatspecifically binds to the EGF Receptor, wherein the primary nucleic acidsequence is selected SEQ ID NOS: 1-54. In other embodiments, the primarynucleic acid sequence is selected such that it is at least about 50%identical, at least about 60% identical, at least about 70% identical,at least about 75% identical, at least about 80% identical, at leastabout 85% identical, at least about 90% identical, or at least about 95%identical to a primary nucleic acid sequence selected from SEQ ID NOS:1-55.

In further embodiments, the present disclosure provides an aptamer thatspecifically binds to the EGF Receptor, wherein the primary nucleic acidsequence includes a sequence of contiguous nucleotides that areidentical to a sequence of contiguous nucleotides included in any of SEQID NOS: 34 to 55. For example, in various embodiments, the sequence ofcontiguous nucleotides in the EGFR aptamer can include a sequence ofcontiguous nucleotides selected from an nucleic acid which is comprisedof at least 6 contiguous nucleotides from the consensus sequence, SEQ IDNO: 55; at least 7 contiguous bases from the consensus sequence; of atleast 8 contiguous bases from the consensus sequence; of at least 9contiguous bases from the consensus sequence; of at least 10 contiguousbases from the consensus sequence; of at least 11 contiguous bases fromthe consensus sequence; of at least 12 contiguous bases from theconsensus sequence; of at least 13 contiguous bases from the consensussequence; of at least 14 contiguous bases from the consensus sequence;and of at least 15 contiguous bases from the consensus sequence.

The EGFR aptamer can contain any number of nucleotides in addition tothe EGFR binding region. In various embodiments, the EGFR aptamer caninclude up to about 100 nucleotides, up to about 95 nucleotides, up toabout 90 nucleotides, up to about 85 nucleotides, up to about 80nucleotides, up to about 75 nucleotides, up to about 70 nucleotides, upto about 65 nucleotides, up to about 60 nucleotides, up to about 55nucleotides, up to about 50 nucleotides, up to about 45 nucleotides, upto about 40 nucleotides, up to about 35 nucleotides, up to about 30nucleotides, up to about 25 nucleotides, and up to about 20 nucleotidesin addition to the binding region.

EXAMPLES Example 1 EGFR Aptamer Construct

A nucleic acid aptamer was isolated that only binds EGFR when it is notbound by either of the therapeutic antibodies cetuximab or panitumumab.The aptamer had the nucleic acid sequence of SEQ ID NO: 1, in which each“N” is the modified deoxyuridine, 5-benzylaminocarbonyl-2′-deoxyuridine.

(SEQ ID NO: 3) ANCGAGGNNGNGGGNCGGANNGNNGGANNCNNNAAGNNGGGAACACCAACCGAGAACG

A modified aptamer was isolated that only binds EGFR when it is notbound by either of the therapeutic antibodies cetuximab or panitumumab,shown in SEQ ID NO: 4, in which “N” represents a modified deoxyuridine(5-benzylaminocarbonyl-2′-deoxyuridine). The K_(d) for EGFR of thisaptamer was 1.23×10⁻¹⁰ M.

(SEQ ID NO: 4) CCACGCTGGGTGGGTCANCGAGGNNGNGGGNCGGANNGNNGGANNCNNNAAGNNGGGAACACCAACCGAGAACG

An aptamer having the nucleic acid sequence of SEQ ID NO: 5, a truncateof SEQ ID NO: 4, was found to have a K_(d) for EGFR of 5.26×10⁻¹⁰ M.

An aptamer having the nucleic acid sequence of SEQ ID NO: 6, the 40-merselected region of SEQ ID NO: 4, also had binding affinity for EGFR.

From the same SELEX pool in which the aptamer having the nucleic acidsequence of SEQ ID NO: 4 was isolated, an aptamer having the nucleicacid sequence of SEQ ID NO: 7 was identified to have binding affinity toEGFR.

The nucleic acid sequence represented by SEQ ID NO: 8 was present inboth the aptamer of SEQ ID NO: 4 and the aptamer of SEQ ID NO: 7,representing a consensus sequence present in the two distinct,independently isolated aptamers.

The aptamers were then further modified with a hexaethylene glycol (HEG)spacer molecule on the 5′ end that connected the aptamer to aphotocleavable biotin molecule. The aptamer was also modified with aninverted deoxythymidine on the 3′ end. A schematic of the aptamerconstruct is shown in FIG. 2.

Example 2 Sample Preparation

Serum samples containing EGFR were added to a 96-well plate. Test wellsreceived either cetuximab (Bristol-Meyer NDC 66733-948-23) orpanitumumab (Amgen NDC 55513-954-01) at varying concentrations (20μg/mL, 10 μg/mL, 5 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.05 μg/mL, control withno antibody). A negative control antibody at 60 μg/mL was also prepared.Serum standards lacking the therapeutic antibodies and with variousconcentrations of exogenous EGFR were also prepared (1000 pM, 200 pM, 40pM, 8 pM, 1.6 pM, 0.32 pM, and 0.06 pM of EGFR). All dilutions ofantibodies, serum, and EGFR standards were made with SB17T buffer.

Example 3 Aptamer-EGFR Complex Formation and Partitioning from Sample

The aptamer of SEQ ID NO: 4 modified as in FIG. 2 was added toantibody-spiked serum samples and standards as described in Example 2 toa final concentration of 1 nM. The 96-well plate was incubated for 3hours at 37° C. to achieve equilibrium binding between EGFR in thesample and the aptamer. The serum sample/aptamer mixture from each wellwas transferred to a streptavidin-coated 96 well plate and incubated for30 minutes at 37° C. with shaking at 400 rpm.

Example 4 Biotin Labeling of EGFR in Complex

Patient samples were prepared, mixed, and partitioned on a 96-well plateas described in Example 3. Following the streptavidin incubation, theplate was washed with PB buffer containing 1 mM dextran sulfate and 500μM biotin to saturate any unbound streptavidin, and then washed withPB1T.

Next, 1 mM NHS-PEG4-Biotin (diluted in PB1T pH 7.25) was added to theplate and incubated for 5 minutes at room temperature while being shakenat 600 rpm. The plate was then washed with 20 mM glycine in PB1T, andthen with PB1T.

Example 5 Partitioning of Aptamer-EGFR Complex from Unbound Aptamer

Patient samples were prepared, mixed, and partitioned from the sample asdescribed in Examples 1-3, and the aptamer-EGFR complex was bound to theplate and the EGFR was biotinylated as described in Example 4. Next, theplate wells were exposed to 1 mM dextran sulfate in PB1 buffer, exposedto UV light, and shaken for 5 minutes while shaking at 600 rpm.

The supernatant was then removed from the plate and added to anotherstreptavidin-coated 96-well plate (previously washed in NaOH) andincubated for 10 minutes at room temperature with shaking. The plate wasthen washed with 50% PG in PB1T buffer, and then in PB buffer.

Example 6 Removal of Aptamer from Plate-Bound EGFR

Patient samples were prepared, mixed, and partitioned from the sample asdescribed in Examples 1-3. The aptamer-EGFR complex was bound to theplate, the EGFR was biotinylated, and the complex was partitioned fromunbound aptamer as described in Examples 4 and 5.

The aptamer was then released from the EGFR by adding CAPSO buffer andincubated for 5 minutes with shaking at 600 rpm. The supernatant wasremoved to a 96-well plate and neutralization buffer was added to thesupernatant to raise the pH.

Example 7 Aptamer Detection and Quantitation

Patient samples were prepared, mixed, and partitioned from the sample asdescribed in Examples 1-3. The aptamer-EGFR complex was bound to theplate and the EGFR was biotinylated, as described in Example 4.Following partitioning of the aptamer-EGFR complex from unbound aptameras described in Example 5, the aptamer was removed from the plate-boundEGFR as described in Example 6.

The aptamer was then detected using a commercially available xTAG® assay(Luminex Corp., Austin, Tex.). Data was generated by SomaLogic®,Boulder, Co., USA.

Results

FIG. 3 shows a graph of the fluorescent intensity from a Luminex xTag®assay for serum samples spiked with successively decreasing amounts ofeither cetuximab or panitumumab. As the concentration of each of thetherapeutic antibodies decreases in the spiked samples, the amount ofaptamer detected increases. These data show that the aptamer binds EGFRthat is not bound to either cetuximab or panitumumab.

Example 8 Development of an Assay Based on SOMAmer Affinity Reagentsthat Detects Drug-Unbound Serum EGFR in the Presence of Cetuximab andPanitumumab

A Slow Off-Rate Modified Aptamer (SOMAmer) targeting the EGFR ECD wasselected via Systematic Evolution of Ligands by Exponential Enrichment(SELEX). Using this SOMAmer as a capturing reagent and based onpublished studies (Gold, et al. PLoS ONE; Dec. 7,2010:10.1371/journal.pone.0015004), we developed a quantitative serumEGFR assay to reliably quantify EGFR in serum.

Recovery tests using various amounts of purified EGFR spiked into serumdemonstrated a full level of EGFR. Intra and inter assay variabilitywere tested and showed minimum variability. The detection range is 0.95ng/ml to 600 ng/ml.

Interestingly, when serum samples from patients taking cetuximab orpanitumumab at the time of blood collection were tested, we observedmarkedly lower levels of EGFR-captured SOMAmer. ELISA assays from 2different vendors showed normal to high levels of EGFR in these samples.We further showed that pretreatment of normal serum with eithercetuximab or panitumumab can dose-dependently reduce the EGFR SOMAmersignal. The data suggest that our EGFR SOMAmer assay detects serum EGFRmolecules that are unbound by cetuximab or panitumumab. Some treatedpatient samples had more drastic reductions in circulating serum EGFRthan others.

Various assay development parameters such as accuracy, detection range,and intra and inter assay variability showed that a SOMAmer-based assaydetecting serum EGFR can be used in a clinical setting. Our data suggestthat this assay can accurately measure drug-unbound EGFR in patients,which may serve as a surrogate drug efficacy indicator, and this mayhelp physicians to adjust the drug dosage.

Example 9 Further Development of an Assay Based on SOMAmer (SlowOff-Rate Modified Aptamer) Affinity Reagents that Detects Drug-UnboundSerum EGFR in the Presence of Cetuximab and Panitumumab

We describe a serum EGFR assay that may provide a means to address thevariability in cetuximab and/or panitumumab interaction with EGFR. Thisassay is based on slow off-rate modified aptamer (SOMAmer™; SomaLogic,Boulder, Colo.) technology (Gold L, Ayers D, Bertino J, et al.Aptamer-based multiplexed proteomic technology for biomarker discovery.PLoS One. 2010; 5:e15004.; Kraemer S, Vaught J D, Bock C, et al. FromSOMAmer-based biomarker discovery to diagnostic and clinicalapplications: a SOMAmer-based, streamlined multiplex proteomic assay.PLoS One; 2011; 6:e26332.), which appears to measure circulating EGFRECD that is not bound by cetuximab or panitumumab.

Patient Samples. Serum samples used for this study were collected fromhealthy individuals and tested individually (EGFR recovery studies) oras pooled sera (drug interference studies).

SOMAmer Assay. SOMAmers are single-stranded DNA molecules (aptamers)containing modified nucleotides that use their unique secondarystructures to create specific and long lasting interactions with theirtarget proteins. SOMAmers are coupled with a biotin moiety via aphoto-cleavable linker sequence.

FIG. 6 describes the work flow of the SOMAmer assay. In brief, dilutedserum is incubated with SOMAmers, and the SOMAmers are captured on astreptavidin plate. Proteins not bound to the SOMAmer (non-EGFRproteins) are washed away, and the captured proteins are labeled withanother biotin moiety. The plate is then exposed to LED light to breakthe link between the biotin and aptamer and releases both protein-boundand unbound aptamers. The supernatant is then transferred to a newstreptavidin plate allowing this time biotinylated protein-streptavidininteraction, washed to remove protein-unbound aptamer, and exposed toalkaline buffer to release the protein-bound aptamer component, whichthen serves as the template for quantitative real-time PCR (qPCR).

EGFR qPCR Assay. We used SYBR® green (Life Technologies, Carlsbad,Calif.)-based qPCR. In addition to the Taq DNA polymerase, we also addedKOD XL polymerase to enhance readthrough of modified nucleotides. TheABI ViiA 7 instrument (Applied Biosystems, Foster City, Calif.) wasused. A typical example of a qPCR using serially diluted EGFR-capturedSOMAmer is shown on FIG. 7.

Purified EGFR protein was serially diluted in 3-fold steps from 600 to2.5 ng/L. Each dilution was further diluted to 30-fold using the SB17Tbuffer to mimic the serum dilution. The EGFR SOMAmer assay and qPCR weredone as described in the Materials and Methods. Triplicate runs wereperformed for each dilution.

EGFR ELISA. The EGFR ELISA assay was performed according to themanufacturer's instructions (Wilex, Cambridge, Mass.).

Recovery studies using serum samples spiked with different levels ofpurified EGFR (30, 150, and 300 ng/mL) demonstrated recovery of 84.0% to119.8% (mean recovery=100.7%; FIG. 8). The average percent coefficientof variation (% CV) was 6.9%. These findings indicate that this SOMAmerassay can accurately detect EGFR ECD in serum of untreated individuals.

We tested interference from 2 widely used EGFR ECD-targeting monoclonalantibody drugs (cetuximab and panitumumab) with both the SOMAmer assayand a commercially available ELISA. Pre-incubation of serum withcetuximab or panitumumab lowered the recovery of EGFR SOMAmer in adose-dependent manner, but did not notably affect ELISA results (FIG.9). These findings suggest that the EGFR SOMAmer detects primarily thedrug-unbound fraction of EGFR in serum containing cetuximab orpanitumumab.

We also performed the SOMAmer EGFR assay on samples from 3 patients withknown treatment history and drug response. The results are summarized inthe Table 1.

TABLE 1 Total and Unbound EGFR Levels in Patients With Metastatic ColonCancer ELISA SOMAmer Total Unbound % EGFR, EGFR, Unbound Drug Patientng/mL ng/mL EGFR Drug Response 1 285 15 5 Panitumumab Partial 2 105 7571 Panitumumab Partial 3 113 6 5 Cetuximab Complete

This SOMAmer-based assay can accurately measure EGFR in serum ofpatients not receiving anti-EGFR therapy. Whereas the EGFR ELISAmeasures drug-bound and unbound EGFR, the EGFR SOMAmer assay detectsprimarily unbound EGFR in patients receiving cetuximab or panitumumabtreatment. High levels of unbound drug could be an indicator of poorinteraction between drug and target.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

That which is claimed is:
 1. A method for determining the amount of freeEpidermal Growth Factor Receptor (EGFR) in a sample comprising: (a)contacting a sample comprising EGFR with an aptamer that specificallybinds to free EGFR under conditions that allow binding of the aptamer tothe free EGFR in the sample to form an aptamer-EGFR complex, wherein thefree EGFR is EGFR that is not bound to a therapeutic molecule if presentin the sample, and wherein the aptamer comprises the structure definedby SEQ ID NO: 55; (b) detecting the amount of the free EGFR bound by theaptamer; and (c) determining the amount of free EGFR in the sample basedon the amount of the free EGFR bound by the aptamer.
 2. The method ofclaim 1, wherein the aptamer comprises the structure defined by asequence selected from the group comprising SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO:
 8. 3. The method of claim 1,wherein the aptamer comprises a first binding pair member and isimmobilized on a first solid support via a second binding pair memberthat specifically binds to the first binding pair member.
 4. The methodof claim 3, wherein the sample is contacted with the aptamer prior toimmobilization on the first solid support.
 5. The method of claim 3,wherein the sample is contacted with the aptamer after immobilization onthe first solid support.
 6. The method of claim 3, wherein the amount offree EGFR is determined by determining the amount of EGFR immobilized onthe first solid support.
 7. The method of claim 6, wherein the amount ofEGFR immobilized on the first solid support is determined using anantibody that specifically binds to the immobilized EGFR.
 8. The methodof claim 3, said method further comprising: (i) attaching a thirdbinding pair member to the EGFR; (ii) releasing the aptamer-EGFR complexfrom the solid support; (iii) immobilizing the aptamer-EGFR complex on asecond solid support via a fourth binding pair member that specificallybinds to the third binding pair member; and (iv) determining the amountof EGFR immobilized to the second solid support.
 9. The method of claim8, wherein the amount of EGFR immobilized on the second solid support isdetermined using an antibody that specifically binds to EGFR.
 10. Themethod of claim 8, said method further comprises labeling the EGFR witha detectable label either before or after immobilization on the secondsolid support.
 11. The method of claim 10, wherein the aptamer furthercomprises a detectable label.
 12. The method of claim 8, wherein theamount of EGFR immobilized on the solid support is determined using flowcytometry.
 13. The method of claim 8, wherein the amount of EGFRimmobilized on the second solid support is determined by releasing theaptamer from the aptamer-EGFR complex and determining the amount of thereleased aptamer.
 14. The method of claim 13, wherein the amount of thereleased aptamer is determined by amplification of the aptamer.
 15. Themethod of claim 1, further comprising detecting the total amount of EGFRin the sample using an enzyme-linked immunosorbent assay.
 16. The methodof claim 1, wherein the sample is a serum sample.
 17. The method ofclaim 1, wherein the sample is from a human patient.
 18. The method ofclaim 17, wherein the patient has cancer.
 19. The method of claim 17,wherein the patient is receiving an anti-EGFR receptor therapy.
 20. Themethod of claim 17, wherein the patient is receiving cetuximab orpanitumumab.
 21. The method of claim 1, wherein the therapeutic moleculeis an anti-EGFR receptor antibody.
 22. The method of claim 21, whereinthe anti-EGFR receptor antibody is cetuximab or panitumumab.
 23. Themethod of claim 1, wherein X of SEQ ID NO: 55 is G, C, or A; Y of SEQ IDNO: 55 is G or A and/or each N of SEQ ID NO: 55 is, independently, a5-position modified 2′-deoxyuridine.
 24. The method of claim 1, whereineach N of SEQ ID NO: 55 is, independently, selected from a5-benzylaminocarbonyl-2′-deoxyuridine,5-(N-benzylcarboxyamide)-2′-deoxyuridine,5-(N-isobutylcarboxyamide)-2′-deoxyuridine,5-(N-tryptaminocarboxyamide)-2′-deoxyuridine,5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridinechloride, 5-(N-naphthylmethylcarboxamide)-2′-deoxyuridine and a5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine.